US5312922A - Europium and terbium chelators for time-resolved fluorometric assays - Google Patents
Europium and terbium chelators for time-resolved fluorometric assays Download PDFInfo
- Publication number
- US5312922A US5312922A US07/863,746 US86374692A US5312922A US 5312922 A US5312922 A US 5312922A US 86374692 A US86374692 A US 86374692A US 5312922 A US5312922 A US 5312922A
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- United States
- Prior art keywords
- chelators
- fluorescent
- sup
- complexes
- fluorescence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 239000002738 chelating agent Substances 0.000 title claims abstract description 55
- 229910052693 Europium Inorganic materials 0.000 title claims description 6
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium atom Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 title claims description 6
- 229910052771 Terbium Inorganic materials 0.000 title claims description 5
- GZCRRIHWUXGPOV-UHFFFAOYSA-N terbium atom Chemical compound [Tb] GZCRRIHWUXGPOV-UHFFFAOYSA-N 0.000 title claims description 5
- 238000007421 fluorometric assay Methods 0.000 title 1
- 229910052747 lanthanoid Inorganic materials 0.000 claims abstract description 12
- 150000002602 lanthanoids Chemical class 0.000 claims abstract description 12
- 150000001875 compounds Chemical class 0.000 claims description 9
- 229910052751 metal Inorganic materials 0.000 claims description 5
- 239000002184 metal Substances 0.000 claims description 5
- 102000002260 Alkaline Phosphatase Human genes 0.000 abstract description 23
- 108020004774 Alkaline Phosphatase Proteins 0.000 abstract description 23
- 239000000758 substrate Substances 0.000 abstract description 23
- 238000003018 immunoassay Methods 0.000 abstract description 22
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 abstract description 11
- HSHNITRMYYLLCV-UHFFFAOYSA-N 4-methylumbelliferone Chemical compound C1=C(O)C=CC2=C1OC(=O)C=C2C HSHNITRMYYLLCV-UHFFFAOYSA-N 0.000 abstract description 10
- 229910019142 PO4 Inorganic materials 0.000 abstract description 8
- 239000010452 phosphate Substances 0.000 abstract description 8
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 abstract description 5
- BCHIXGBGRHLSBE-UHFFFAOYSA-N (4-methyl-2-oxochromen-7-yl) dihydrogen phosphate Chemical compound C1=C(OP(O)(O)=O)C=CC2=C1OC(=O)C=C2C BCHIXGBGRHLSBE-UHFFFAOYSA-N 0.000 abstract description 4
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- 108010061174 Thyrotropin Proteins 0.000 abstract description 3
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- -1 phosphate ester Chemical class 0.000 description 3
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- DXALAFAFIXJDOS-UHFFFAOYSA-N 4-bromo-2,8-bis(trifluoromethyl)quinoline Chemical compound C1=CC=C(C(F)(F)F)C2=NC(C(F)(F)F)=CC(Br)=C21 DXALAFAFIXJDOS-UHFFFAOYSA-N 0.000 description 2
- HDHQZCHIXUUSMK-UHFFFAOYSA-N 4-hydroxy-2-quinolone Chemical compound C1=CC=C2C(O)=CC(=O)NC2=C1 HDHQZCHIXUUSMK-UHFFFAOYSA-N 0.000 description 2
- BIMHWDJKNOMNLD-UHFFFAOYSA-N 5-Hydroxyindole-2-carboxylic acid Chemical compound OC1=CC=C2NC(C(=O)O)=CC2=C1 BIMHWDJKNOMNLD-UHFFFAOYSA-N 0.000 description 2
- PPDRLQLKHRZIJC-UHFFFAOYSA-N 5-nitrosalicylic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC=C1O PPDRLQLKHRZIJC-UHFFFAOYSA-N 0.000 description 2
- BLHCMGRVFXRYRN-UHFFFAOYSA-N 6-hydroxynicotinic acid Chemical compound OC(=O)C1=CC=C(O)N=C1 BLHCMGRVFXRYRN-UHFFFAOYSA-N 0.000 description 2
- XRIHTJYXIHOBDQ-UHFFFAOYSA-N 6-methyl-2-oxo-1h-pyridine-3-carboxylic acid Chemical compound CC1=CC=C(C(O)=O)C(=O)N1 XRIHTJYXIHOBDQ-UHFFFAOYSA-N 0.000 description 2
- WHJTTWIMQIGVJK-UHFFFAOYSA-N 7-methyl-4-oxo-1h-1,8-naphthyridine-3-carboxylic acid Chemical compound N1C=C(C(O)=O)C(=O)C=2C1=NC(C)=CC=2 WHJTTWIMQIGVJK-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- GXAMYUGOODKVRM-UHFFFAOYSA-N Flurecol Chemical compound C1=CC=C2C(C(=O)O)(O)C3=CC=CC=C3C2=C1 GXAMYUGOODKVRM-UHFFFAOYSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
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- 239000002253 acid Substances 0.000 description 2
- 239000012670 alkaline solution Substances 0.000 description 2
- 229940027991 antiseptic and disinfectant quinoline derivative Drugs 0.000 description 2
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- 238000003776 cleavage reaction Methods 0.000 description 2
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- 238000013461 design Methods 0.000 description 2
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- 238000010790 dilution Methods 0.000 description 2
- KAKKHKRHCKCAGH-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate;hexahydrate Chemical compound O.O.O.O.O.O.[Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 KAKKHKRHCKCAGH-UHFFFAOYSA-L 0.000 description 2
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- 230000036963 noncompetitive effect Effects 0.000 description 2
- DJXNJVFEFSWHLY-UHFFFAOYSA-N quinoline-3-carboxylic acid Chemical compound C1=CC=CC2=CC(C(=O)O)=CN=C21 DJXNJVFEFSWHLY-UHFFFAOYSA-N 0.000 description 2
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- FBZONXHGGPHHIY-UHFFFAOYSA-N xanthurenic acid Chemical compound C1=CC=C(O)C2=NC(C(=O)O)=CC(O)=C21 FBZONXHGGPHHIY-UHFFFAOYSA-N 0.000 description 2
- XVSWBEIQRFTJKH-UHFFFAOYSA-N (5-fluoro-2-hydroxyphenyl)methyl dihydrogen phosphate Chemical compound OC1=CC=C(F)C=C1COP(O)(O)=O XVSWBEIQRFTJKH-UHFFFAOYSA-N 0.000 description 1
- JYTNCILGSJWIDM-UHFFFAOYSA-N 1,3-dihydroxyacridine-9-carboxylic acid Chemical compound OC1=CC(O)=C2C(C(=O)O)=C(C=CC=C3)C3=NC2=C1 JYTNCILGSJWIDM-UHFFFAOYSA-N 0.000 description 1
- DRGAZIDRYFYHIJ-UHFFFAOYSA-N 2,2':6',2''-terpyridine Chemical group N1=CC=CC=C1C1=CC=CC(C=2N=CC=CC=2)=N1 DRGAZIDRYFYHIJ-UHFFFAOYSA-N 0.000 description 1
- LWVQRKXAZCLJPX-UHFFFAOYSA-N 2-(3-hydroxy-4,7,7-trimethyl-2-bicyclo[2.2.1]heptanyl)acetic acid Chemical compound C1CC2(C)C(O)C(CC(O)=O)C1C2(C)C LWVQRKXAZCLJPX-UHFFFAOYSA-N 0.000 description 1
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- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
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- PKWIYNIDEDLDCJ-UHFFFAOYSA-N guanazole Chemical compound NC1=NNC(N)=N1 PKWIYNIDEDLDCJ-UHFFFAOYSA-N 0.000 description 1
- 125000005843 halogen group Chemical group 0.000 description 1
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- SYJXFKPQNSDJLI-HKEUSBCWSA-N neamine Chemical compound N[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](N)C[C@@H]1N SYJXFKPQNSDJLI-HKEUSBCWSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
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- 108020004707 nucleic acids Proteins 0.000 description 1
- 239000013110 organic ligand Substances 0.000 description 1
- 229940083251 peripheral vasodilators purine derivative Drugs 0.000 description 1
- 150000005041 phenanthrolines Chemical class 0.000 description 1
- 150000003014 phosphoric acid esters Chemical class 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
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- 125000000561 purinyl group Chemical class N1=C(N=C2N=CNC2=C1)* 0.000 description 1
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- FFRYUAVNPBUEIC-UHFFFAOYSA-N quinoxalin-2-ol Chemical compound C1=CC=CC2=NC(O)=CN=C21 FFRYUAVNPBUEIC-UHFFFAOYSA-N 0.000 description 1
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D213/79—Acids; Esters
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
- C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
- C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D215/48—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
- C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
- C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D215/48—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
- C07D215/54—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen attached in position 3
- C07D215/56—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen attached in position 3 with oxygen atoms in position 4
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
- C07D241/36—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings condensed with carbocyclic rings or ring systems
- C07D241/38—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings condensed with carbocyclic rings or ring systems with only hydrogen or carbon atoms directly attached to the ring nitrogen atoms
- C07D241/40—Benzopyrazines
- C07D241/44—Benzopyrazines with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the hetero ring
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- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
- C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
- C07D311/16—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted in position 7
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- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/003—Compounds containing elements of Groups 3 or 13 of the Periodic Table without C-Metal linkages
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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- C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
- C12Q2334/20—Coumarin derivatives
- C12Q2334/22—4-Methylumbelliferyl, i.e. beta-methylumbelliferone, 4MU
Definitions
- This invention relates to florescent lanthanide metal chelates.
- Fluorescent lanthanide metal chelates and in particular those for europium and terbium chelates have significant commercial application because of their use as labels in highly sensitive time-resolved fluorometric immunoassays (1-4).
- the first commercially available time-resolved fluorescence immunoassay system, DelfiaTM (available by Pharmacia-LKB, Sweden) uses Eu 3+ as immunological label (5,6).
- a second-generation time-resolved fluorometric immunoassay system uses the europium chelator 4,7-bis (chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) as immunological label (7.8).
- BCPDA europium chelator 4,7-bis (chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid
- Tb 3+ and its chelates have been used as immunological and nucleic acid labels (9,10).
- Tb 3+ is inferior to Eu 3+ in terms of detectability.
- Eu 3+ and Tb 3+ can be used simultaneously for dual analyte assays (11).
- time-resolved fluorometric immunoassays it is desirable to use a chelate-label which can be detected down to the subpicomolar range (5).
- multiple labelling strategies can be used in order to achieve subplcomolar analyte sensitivity (1 and 4-16).
- the primary immunological label is alkaline phosphatase (ALP); its acid (FSAP).
- ALP alkaline phosphatase
- FSAP nonhydrolysed ester
- FSA hydrolysed ester
- FSA When FSA is added to an aqueous alkaline solution containing Tb 3+ -EDTA, a mixed complex is formed which emits long-lived fluorescence characteristic of Tb 3+ .
- FSA is an appropriate ligand for energy transfer to the metal-ion.
- An intact hydroxyl group on the FSA molecule is essential for these highly fluorescent mixed complexes to be formed.
- FSAP does not form any fluorescent complexes with Tb 3+ -EDTA.
- ALP activity can be monitored with use of FSAP as substrate, by measuring the released FSA after adding an alkaline solution of Tb 3+ -EDTA (17). This system has been used for the highly sensitive and rapid quantification of alpha-fetoprotein and thyrotropin in serum (18,19).
- fluorescent lanthanide metal chelates are selected from compounds of the following formulas I through XXXIII: ##STR1##
- the selected esters of chelates of formulas I through XXXIII are enzymatically treated to render the chelates either fluorescent or non-fluorescent when combined with a selected lanthanide metal.
- FIG. 1 Logarithmic plots of fluorescence vs concentration of candidate fluorogenic lanthanide chelator. In all cases A to E but D, the fluorescence of each Eu3+ chelate was higher or equal (C) to the corresponding fluorescence of the Tb3+ chelate; the Eu3+-chelate fluorescence curve in plots A to E is thus on top of the Tb3+-chelate fluorescence curve, except in case D.
- the chelators represented are XI (A, the third curve representing the lowest fluorescence is due to the Tb3+-EDTA-fluorosalicylate complex as described in (17); XIII (B); IX (C); XXIX (D); XXXIII (E); II (F, with Eu3+); XIV (G, with Eu3+); XIX (H, with Eu3+); XXXI (I, lower curve with Eu3+); XXXII (I, upper curve with Eu3+).
- FIG. 2 Calibration curves for the proposed TSH assay (A). Fluorescence vs TSH concentration on linear axes (B). A percentage plot, where Bo is the fluorescence of the zero standard and B the fluorescence of all other standards. The concentration of the substrate used was (in mol/L from bottom to top): 5 ⁇ 10 -6 ; 1 ⁇ 10 -5 ; 2 ⁇ 10 -5 ; 5 ⁇ 10 -5 .
- FIG. 3 Calibration curves for the proposed thyroxine assay (A). Double linear plots. (B) Double logarithmic plots. The substrate concentration and antibody dilutions used were: 5 ⁇ 10 -6 M and 10-fold (1); 5 ⁇ 10 -6 M and 20-fold (2); 2 ⁇ 10 -5 M and 10-fold (3); 2 ⁇ 10 -5 M and 20-fold (4).
- FIG. 4 Correlation between serum thyroxine results obtained with a radioimmunoassay (RIA) and the proposed assay method (TR-FIA).
- RIA radioimmunoassay
- TR-FIA proposed assay method
- the molecules are identified as follows in formulas I through XXXIII: ##STR2##
- the registry numbers for these compounds is as follows: I,3-hydroxyflavone, 577-85-5; II, 4-hydroxyquinoline-2-carboxylic acid, 492-27-3; III, 2-hydroxynicotinic acid, 609-71-2; IV, 1-hydroxy-2-naphthoic acid, 86-48-6; V, 5-fluorosalicyclic acid, 345-16-4; VI, 6-hydroxynicotinic acid, 5006-66-6; VII, 2-hydroxy-6-methylpyridine-3-carboxylic acid, 38116-61-9; VIII, 3-hydroxy-2-naphthoic acid, 92-70-6; IX, 3-hydroxypicolinic acid, 874-24-8; X, 2-hydroxy-5-nitrobenzoic acid, 96-97-9; XI, 4-hydroxy-7-trifluromethyl-3-quinoline carboxylic acid, 574-92-5; XII, 3-hydroxy-4,7
- Molecules IX, XI, XIII, XXIX and XXIII of the above formulae are good fluorogenic chelators for both Eu 3+ and Tb 3+ and molecules IX, XI, XIII, XIV, XXIX and XXXII of the above formulae are good fluorogenic chelators for Eu 3+ only. Under optimised measuring conditions of pH and presence or absence of EDTA, these chelators can be detected at levels ⁇ 10 -8 M. Thus, these substrates demonstrate utility in highly sensitive enzymatically amplified time-resolved fluorometric immunoassays.
- the candidate organic chelators tested are shown in FIG. 1. All of them were obtained from Aldrich Chemical Co., Milwaukee, Wis. 53233. Calf intestine alkaline phosphatase (ALP) was obtained from Boehringer Mannheim Canada, Montreal PQ. Alkaline phosphatase-labelled streptavidin (SA-ALP) was obtained from Zymed Laboratories Inc., San Fransisco, Calif. 94080, as a 0.75 mg/mL solution. White, opaque 12-well polystyrene microtiter strips coated with monoclonal anti-thyrotropin antibody (for the TSH assay) or a thyroxine-globulin conjugate (for the T4 assay) were obtained from CyberFluor.
- biotinylated detection antibodies, standards and buffers needed for the immunoassays were also from CyberFluor.
- Europium (III) chloride hexahydrate and terbium (III) chloride hexahydrate were from Aldrich. All other chemicals were from Sigma Chemical Co., St. Louis, Mo. 63178.
- the alkaline phosphatase substrate buffer was a 0.1 mol/L Tris, pH 9.0, containing 0.1 mol/L NaCl and 1 mmol/L MgCl 2 .
- the diluent for the biotinylated detection TSH antibody and the SA-ALP conjugate was a 6% (w/v) solution of bovine serum albumin (BSA) in a 50 mmol/L Tris buffer, pH 7.40, containing 0.5 g of sodium azide per liter.
- the wash solution was prepared by dissolving 9 g of NaCl and 0.5 mL of polyoxyethylenesorbltan monolaurate (Tween 20) in 1 liter of distilled water.
- All chelators were screened by mixing 100 ⁇ L of a chelator solution and 100 ⁇ L of a Eu 3+ or Tb 3+ solution, (4 ⁇ 10 -3 mol/L in water) and measuring the delayed fluorescence on the 615TM Immunoanalyzer.
- the chelators according to preferred embodiments of the invention were further tested with Eu 3+ or Tb 3+ solutions of various pH, in the absence or presence of EDTA.
- Calibration curves and detection limits were constructed or calculated by adding 100 ⁇ L of an aqueous Eu 3+ or Tb 3+ solution to 100 ⁇ L of a chelator solution in a 0.1 mol/L Tris buffer of pH 9.0.
- chelator according to formula V we added 100 ⁇ L of a Tb 3+ -EDTA solution, 10 -3 mol/L in a 0.5 mol/L Tris buffer (18) of pH 12.5.
- chelators of formula XI and XIII we added 100 ⁇ L of a 4 ⁇ 10-3 mol/L solution of Eu 3+ or Tb 3+ in a 0.1 mol/L Tris buffer, pH 11, containing 5 ⁇ 10 -3 mol/L EDTA.
- chelators of the formulae possess a hydroxyl group which could be converted to a phosphate ester or galactoside for the purpose of using them as substrates of alkaline phosphatase or ⁇ -galactosidase as has already been reported for the chelator of formula V which is included for comparison (17-19).
- sets of candidate chelators with only one group difference were tested i.e. chelators of formualae XIV, XX and XXXI, to demonstrate the importance of the different groups.
- Derivatives of a particular structure i.e.
- chelators Two chelators, XI and XIII exhibited the property of working in the presence of excess EDTA i.e. they form fluorescent complexes with Eu 3+ and Tb 3+ chelated to EDTA. This property is also seen with chelator V (previously reported). In all other cases, fluorescence of the complexes decreases dramatically in the presence of EDTA. For all the chelators, the fluorescence measured is long-lived and lanthanide-specific. Native chelator fluorescence may also exist but is not detected by the time-resolved fluorometer.
- Fluorescence intensity of the complexes varies with pH as shown in Table 1.
- solutions of Eu 3+ and Tb 3+ in buffers of alkaline pH deteriorate quickly due to hydroxide precipitation.
- complexes are preferably formed with the addition of an aqueous solution of Eu 3+ or Tb 3+ (pH ⁇ 6.5) which is stable for long periods of time at room temperature.
- the Eu 3+ and Tb 3+ solutions were made in the presence of excess EDTA at pH 12.5 (for formula V) or 11, respectively. These solutions were also very stable when stored at room temperature.
- the fluorescence intensity of the complexes was highest when the Eu 3+ and Tb 3+ concentration in the final solution was around 2 ⁇ 10 -3 mol/L. Lower concentrations resulted in lower fluorescence signals.
- Fluorescence intensity remained stable ( ⁇ 10%) for at least 3 h in the case of chelators IX, XI, XIII, XIV, XXIX and XXXII and Eu 3+ and/or Tb 3+ .
- Fluorescence intensity decreased with time (change ⁇ 10% per hour) in the case of chelators II, XIX, XXXI and XXXIII. This decrease was also noticed for chelator V and previously reported (18).
- the modification of the structures of these chelators i.e. tranformation into phosphate esters or galactosides can provide desired spectroscopic changes and/or chelation changes and/or energy transfer changes, the net result being an increase or loss of fluorescence in the presence of the lanthanides depending on the desired effect.
- substitution of various entities of the compounds of formulae I to XXIII may be done to enhance performance. Such substitutions may be with existing radicals or at other positions about the ringed structure. Such substition may be in regard to other halogen groups, other alkyl groups and other alkylene groups and the like.
- Another interesting application of the invention is to have a pair of compounds A and B one of which, A, is an enzyme substrate that forms fluorescent complexes with Eu 3+ or Tb 3+ .
- A is an enzyme substrate that forms fluorescent complexes with Eu 3+ or Tb 3+ .
- XXXIII is a non-fluorescent ALP substrate; when ALP cleaves-off phosphate, the product, XXVI, exhibits blue, short-lived fluorescence.
- the pair XXXIII, XXVI and Eu 3+ were used to develop two model time-resolved fluorometric immunoassays for TSH and T4 in serum.
- the design of the TSH assay is of the non-competitive heterogeneous immunoassay format where an immunocomplex of the type solid-phase-coating antibody-TSH-detection antibody-biotin-streptavidin-alkaline phosphatase, is formed in polystyrene microtiter wells.
- the activity of ALP can then be measured in a number of different conventional ways. In this work, we detected ALP by adding the substrate XXXIII for 30 min, followed by addition of a 4 ⁇ 1 0 -3 mol/L Eu 3+ solution to form the fluorescent complex with the unreacted substrate. The calibration curves obtained are shown in FIG. 2. The sensitivity and range of assay can be adjusted by adjusting the concentration of the substrate added.
- T4 a competitive assay for T4 was designed as follows: Polystyrene microtiter wells were coated with a T4-protein conjugate as described previously (20). In the assay, T4 in the sample competes with immobilized T4 for binding to a biotinylated anti-T4 monclonal antibody. After washing out excess reagents, we added a SA-ALP conjugate to form the immunocomplex: solid-phase-T4-antibodybiotin-SA-ALP. The activity of ALP can then be quantified with use of the substrate, XXXIII.
- the amount of signal generated by ALP is inversely related to the analyte concentration and the calibration curves have a shape similar to those of FIG. 2.
- Calibration curves obtained with the system are shown in FIG. 3. Sensitivity can be adjusted by changing the antibody dilution, the substrate concentration and the substrate incubation time. Under optimised conditions, 30 clinical samples have been analyzed by the method of this invention and by a conventional radioimmunoassay procedure. The comparison is shown in FIG. 4. A good correlation exists between the two methods.
- Enzymatically amplified time-resolved fluorescence immunoassays exhibit excellent sensitivity and can be used for the routine manual or automated assay of many analytes in biological fluids.
- the first system reported is based on alkaline phosphatase and the substrate 5-fluorosalicyl phosphate (FSAP) in combination with Tb 3+ (17-19).
- FSAP 5-fluorosalicyl phosphate
- Other possible chelators can be used which offer better detectability in comparison to FSA.
- some of these chelators form stable complexes with Eu 3+ and Tb 3+ thus being advantageous to the previously reported system in which fluorescence decays with time.
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US07/863,746 US5312922A (en) | 1992-04-06 | 1992-04-06 | Europium and terbium chelators for time-resolved fluorometric assays |
AT93907728T ATE185801T1 (de) | 1992-04-06 | 1993-04-06 | Europium und terbium chelatore für zeitgelöste fluorometrische teste |
DE69326834T DE69326834T2 (de) | 1992-04-06 | 1993-04-06 | Europium und terbium chelatore für zeitgelöste fluorometrische teste |
AU38853/93A AU3885393A (en) | 1992-04-06 | 1993-04-06 | Europium and terbium chelators for time-resolved fluorometric assays |
EP93907728A EP0635006B1 (de) | 1992-04-06 | 1993-04-06 | Europium und terbium chelatore für zeitgelöste fluorometrische teste |
PCT/CA1993/000153 WO1993020054A1 (en) | 1992-04-06 | 1993-04-06 | Europium and terbium chelators for time-resolved fluorometric assays |
US08/313,300 US5854008A (en) | 1992-04-06 | 1993-04-06 | Europium and terbium chelators for the time-resolved fluorometric assays |
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Also Published As
Publication number | Publication date |
---|---|
DE69326834T2 (de) | 2000-04-27 |
AU3885393A (en) | 1993-11-08 |
US5854008A (en) | 1998-12-29 |
ATE185801T1 (de) | 1999-11-15 |
EP0635006A1 (de) | 1995-01-25 |
EP0635006B1 (de) | 1999-10-20 |
WO1993020054A1 (en) | 1993-10-14 |
DE69326834D1 (de) | 1999-11-25 |
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